Protein and peptide therapeutic agents comprise an increasing share of the pharmaceutical market with biological drugs making up about $4.5 billion in U.S. sales in 2008. Among these biological drugs are monoclonal antibodies, hormones, and therapeutic enzymes.
Despite the growth of the biopharmaceutical market, implementation of therapeutic proteins remains a challenging task. The inherent physical and chemical instability of proteins can lead to conformational changes, degradation, aggregation, precipitation, and adsorption onto surfaces, each of which can diminish the protein's activity or render it completely inactive.
Once administered, therapeutic proteins are susceptible to short half lives, proteolysis, opsonization, and can cause immunogenic responses, each resulting in undesirable pharmacokinetics. A variety of technologies have been developed to improve these shortfalls. Included among these technologies are amino acid manipulation, genetic fusion of immunoglobulin domains or serum proteins, and conjugation with natural and synthetic polymers.
One successful implementation is the covalent conjugation of the therapeutic protein with polyethylene glycol (PEG), a non-toxic, non-immunogenetic polymer. The process of conjugation of PEG to the therapeutic protein is commonly referred to as PEGylation. PEGylation is known to change the physical and chemical properties of the biomolecule, including conformation, electrostatic binding, and hydrophobicity, and can result in improved pharmacokinetic properties for the drug. Advantages of PEGylation include improvements in drug solubility and diminution of immunogenicity, increased drug stability and circulatory life once administered, and reductions in proteolysis and renal excretion, which allows for reduced frequency of dosing.
PEGylation technology has been advantageously applied to therapeutic proteins and oligonucleotides to provide new drugs that have been approved by the U.S. FDA: adensosine deaminase (Pegademenase), asparaginase (Pegaspargase), G-CSF (Pegfilgrastim), interferon-α2a (Peginterferon-α2a), interferon-α2b (Peginterferon-α2b), hGH (Pegvisomant), anti-VEGF aptamer (Pegaptanib), erythropoietin (PEG-EPO), and anti-TNFα Fab′ (Certolizumab).
PEGylated therapeutic enzymes presently approved and on the market include depleting enzymes that are used to eliminate certain amino acids from the blood stream to starve growing tumor cells. Examples include PEG-asparaginase, PEG-methioninase, and PEG-arginine deiminase. For these enzymes, their relatively small amino acid substrates are able to diffuse through the PEG layer, enter the active site of the enzyme, and are effectively disposed of as designed. However, biomolecules requiring polymeric protection and that act on larger substrates have not been able to be developed because of the required level of PEGylation to retain the enzyme in the body, which renders the active site inaccessible.
PEGylation and PEGylated biomolecules are not without their drawbacks. Due to its strong attraction to water, PEG is known to take on a hydrodynamic diameter significantly greater than would be predicted by their molecular weight. This ballooning effect can be attributed to the reduction in the ability of the body to recognize the biomolecule and results in a decrease in the biomolecule's activity.
A murine A7 monoclonal antibody PEGylated with ten (10) 5 kDa PEG has an activity that is 10% that of the unmodified antibody. Similarly, an interferon-α2a PEGylated with a single 40 kDa PEG has an activity that is 7% that of the unmodified protein. Clearly, modification or hindrance of a biomolecule's active site resulting from PEGylated is a serious problem to be avoided in the development of a biopharmaceutical product.
Despite the advancement in biopharmaceutical drug development provided by PEGylation, a need exists to further advance biopharmaceutical drug development, to overcome the drawbacks associated with PEGylated drugs, and to provide improved reagent and biopharmaceutical products. The present invention seeks to fulfill this need and provides further related advantages.